The goal of my laboratory is to determine how gene expression is regulated in chlamydiae with the long term objective of understanding how chlamydiae cause latent, persistent, and chronic infections. The field of chlamydial research is severely handicapped by the lack of tools to genetically manipulate chlamydiae. However, the development of host-free (intact chlamydiae isolated from host cells) and in vitro transcription methodologies and the successful DNA sequencing of several chlamydial genomes offers new opportunities to identify regulatory DNA elements and transcriptional regulators that bind to these elements. Host-free and in vitro strategies will be used to accomplish four specific aims. 1. Determine the role of alternative sigma factors in gene expression in Chlamydia trachomatis. The temporal expression and the interaction of the sigma factors and their regulators will be characterized, and the promoters regulated by the sigma factors will be identified. 2. Determine the role of integration host factor (IHF) and histones in late gene expression. The genomic DNA binding sites of IHF and the chlamydial histone proteins will be determined, and the effect of histones and IHF on CRP-operon expression in vitro will be examined. 3. Characterize post translational modifications of chlamydial histone proteins. Transmethylation of histones by the chlamydial SET protein will be assayed in vitro with recombinant proteins, and the ability of modified histones to bind to chlamydial DNA will be determined. 4. Determine the pattern of gene expression during the developmental cycle, making use of host-free probes. A microarray consisting of all 894 chromosome and the 8 plasmid ORFs of C. trachomatis D will be probed directly with RNA synthesized by host-free chlamydiae at different times post infection. Emphasis will be placed on transcription at very early times post infection (pi).